Method of cancer treatment by p53 protein control

ABSTRACT

Method of cancer treatment by controlling cellular p53 protein levels. The invention concerns, in particular, the use of a compound capable of modulating calpaine activity.

[0001] The present invention relates to a new method for the treatmentof cancer. More particularly, it relates to a method of treating cancerby regulating the cellular levels of the p53 protein. It also relates tovectors for gene therapy which make it possible to regulate the p53protein, as well as the pharmaceutical compositions containing them.

[0002] For the past fifteen years, the molecular characterization ofoncogenes and of tumour suppressor genes has made it possible to viewthe process of carcinogenesis in a new light. Thus, the increasinglydetailed knowledge of the regulation of these genes and of the functionof the corresponding proteins makes it possible to conceive newtherapeutic approaches.

[0003] More particularly, the elucidation of the breakdown of theoncogenic and anti-oncogenic proteins represents a major challenge interms of the fight against cancer since it presages, in the case ofoncogenic proteins, the possibility of accelerating their degradationand therefore of annihilating their action, in the case of tumoursuppressors, inhibiting their degradation and therefore increasing theirantiproliferative or anti-tumour effect, in the case or mutatedproteins, potentiating their antigenic presentation by molecules of theMajor Histocompatibility Complex and thereby stimulating atumour-specific immune response, and, in the case where the highexpression of the oncogene or of the anti-oncogene is capable ofinducing programmed cell death, the possibility of stabilizing theseproteins so as to trigger the apoptotic process.

[0004] Originally, the p53 protein was classified as a nuclear oncogenesince it could, in transfection experiments, extend the life of rodentcells in culture as well as cooperate with activated oncogenes such asras to transform cells in primary culture. Indeed, the genes used inthese first experiments were mutated and led to the expression ofvariant p53 proteins characterized by a gain in function. Withoutexcluding functions which might still be discovered, it is now knownthat the p53 protein, at least in its wild-type form, is a transcriptionfactor which negatively regulates growth and cell division and which, incertain situations, is capable of inducing apoptosis (Yonish-Rouach etal., Nature, 352, 345-347, 1991). Given that these properties manifestthemselves in a stress situation where the integrity of the cellular DNAis threatened, it has been suggested that p53 is a “guardian of thegenome”. The presence of mutated p53 proteins in about 40% of humantumours, all types taken together, reinforces this hypothesis andunderlines the probably critical role which mutations of this gene playin the tumour development (for reviews, see Montenarh, Oncogene, 7,1673-1680, 1992; Oren, FASEB J., 6, 3169-3176, 1992; Zambetti andLevine, FASEB J., 7, 855-865, 1993).

[0005] The wild-type p53 protein is subject to a complex regulationwhich involves the control of its synthesis and of its breakdown as wellas that of its intracellular location and of its post translationalmodifications (see the reviews cited above). The wild-type p53 proteinis extremely unstable with a half-life of a few minutes. In contrast,some mutated proteins which accumulate at a high level in tumours have asignificantly extended half-life. Little has been clearly established asregards the degradation of p53. Indeed, neither the intracellular sitesof degradation, nor the number and the nature of the catabolic pathwaystaken, nor the peptide units labelling p53 for its degradation areknown. To our knowledge, the only information available relates to theinvolvement of the enzyme E1 of the ubiquitin cycle under certainexperimental conditions (Ciechanover et al., Proc. Natl. Acad. Sci. USA88, 139-143, 1991; Chowdary et al., Molec. Cell. Biol. 14, 1997-2003,1994). Moreover, it has been shown that certain proteolytic productsderived from p53 may be presented in an antigenic manner.

[0006] The present invention results partly from the demonstration thatthe p53 proteins are substrates for calcium-dependent proteases: thecalpains. It results more particularly from the demonstration that thep53 proteins are degraded specifically by m-calpain or μ-calpain. Thepresent invention constitutes the first demonstration of a mechanism forregulating the cellular levels of the p53 proteins and thus offers a newparticularly effective and specific approach for modulating the levelsof this protein in pathological situations such as especially certaincancers.

[0007] In particular, the present invention describes a new approach forthe treatment of cancer, based on the use of compounds which modulatethe activity of calpains on the p53 proteins, which make it possibleeither to activate the degradation of the mutated p53 proteins, in orderto block their tumorigenic effect and/or to enhance the presentation ofimmunogenic peptides, or to stabilize the wild-type p53 protein, inorder to counterbalance the tumorigenic effect of the mutated proteinsexpressed in the tumours and/or in order to induce the apoptosis of thetumour cells.

[0008] A first subject of the invention therefore consists in the use ofa compound capable of modulating the activity of calpain for thepreparation of a pharmaceutical composition for the treatment ofcancers.

[0009] Calpains are ubiquitous enzymes found in most mammalian cells(for a review, see Croall and deMartino, Physiol. Rev., 71, 813-847,1991). They are essentially cytoplasmic but they can penetrate into thenucleus by virtue of the destruction of the nuclear envelope duringmitosis or following certain stimuli. As indicated above, theproteolytic activity of calpains is dependent on the presence ofcalcium.

[0010] The compounds capable of modulating the activity of calpain forthe purposes of the present invention may be of several types.

[0011] They may be compounds capable of inhibiting the activity of thecalpain on the p53 proteins. These compounds are particularlyadvantageous since they can be used to inhibit, at least in part, thedegradation of the wild-type p53 protein. These compounds therefore makeit possible to stabilize intracellularly the wild-type p53 protein andto counterbalance the effect of the mutated forms. Among the inhibitorycompounds which can be used within the framework of the invention theremay be mentioned the protease inhibitors (leupeptin, aprotinin, PMSF,and the like), the calcium chelators (EGTA, EDTA, and the like) or morespecific inhibitors such as calpastatin or any fragment or derivativethereof. Calpastatin is a known inhibitor of the calpains. Its sequencehas been described in the prior art (SEQ ID No. 1). A particularlyadvantageous embodiment of the present invention consists intransferring into the tumours a vector carrying all or part of thesequence encoding calpastatin. This approach is particularly adapted tothe treatment of cancers which always have a wild-type p53 allele, suchas colic or bronchial carcinomas for example. Various fragments orderivatives of calpastatin can be used within the framework of thepresent invention. Such fragments or derivatives may be any moleculeobtained from the sequence SEQ ID No. 1 by modification(s) of a geneticand/or chemical nature, preserving the capacity to inhibit, at least inpart, the activity of a calpain. Modification of a genetic and/orchemical nature is understood to mean any mutation, deletion,substitution, addition and/or modification of one or more nucleotides.Such modifications may be carried out with various ends, especially thatof preparing sequences adapted to expression in a specific type ofvector or host, that of reducing the size of the sequence so as tofacilitate their cellular penetration, that of increasing the inhibitoryactivity, or, in a particularly advantageous manner, of increasing theselectivity of the inhibitor towards the activity of the calpains on thedegradation of the wild-type p53 protein.

[0012] Such modifications may be carried out, for example, by in vitromutagenesis, by introduction of additional constituents or of syntheticsequences, or by deletions or substitutions of the originalconstituents. When a derivative as defined above is prepared, itsactivity as inhibitor of the activity of the, calpains on p53 proteinscan be demonstrated in several ways, and in particular by bringing intocontact the said inhibitor and the various forms of p53 proteins, andthen by detecting the degradation products obtained (see Examples 1 to3). Any other technique known to persons skilled in the art canobviously be used to this effect.

[0013] In a specific embodiment of the present invention, all or part ofcalpastatin, or a nucleic acid encoding all or part of calpastatin isused as inhibitor. Still more particularly, a peptide comprising all orpart of the sequence SEQ ID No. 1 or of a derivative thereof is used.

[0014] As regards more particularly the derivatives, there may bementioned, by way of example, the compound of sequence SEQ ID No. 2,which corresponds to a fragment of calpastatin. There is advantageouslyused any derivative composed of the sequence SEQ ID No. 1 or 2 which iscapable of specifically or preferentially inhibiting the degradation ofthe wild-type p53 protein by calpain.

[0015] The compounds capable of modulating the activity of calpain onthe p53 proteins for the purposes of the present invention may also bederivative of calpain capable of specifically or preferentiallydegrading the mutated p53 proteins. Such derivatives are also veryadvantageous since they make it possible to activate the degradation ofthe mutated p53 proteins, in order to block their tumorigenic effectand/or to increase the presentation of the immunogenic peptides, withoutsignificantly affecting the cellular levels of the wild-type p53protein. Such derivatives may be obtained from calpain, by structuralmodification(s) of a genetic and/or chemical nature. The capacity of thederivatives thus obtained to specifically or preferentially degrade themutated p53 proteins may then be demonstrated as described in Examples 1to 3.

[0016] Preferably, the modulators used within the framework of theinvention are proteins or polypeptides, or nucleic acid sequencesencoding these polypeptides or proteins. Still more preferably, themodulatory compounds are proteins or polypeptides which are specificinhibitors of the activity of calpain on the wild-type p53 protein orforms of calpains, modified or otherwise, for specifically degrading themutated p53 proteins.

[0017] In a particularly advantageous manner, the invention consists inthe possibility of bringing about the expression in cancer cells havingboth a wild-type p53 allele and a mutated p53 allele of nucleicsequences encoding inhibitors of calpain, such as calpastatin or part ofcalpastatin, or forms of calpains, modified or otherwise, forspecifically degrading the mutated p53 proteins.

[0018] The nucleic acid sequence used within the framework of thepresent invention may be administered as such, in the form of naked DNAaccording to the technique described in Application WO 90/11092. It canalso be administered in a form complexed, for example, with DEAE-dextran(Pagano et al., J. Virol. 1 (1967) 891), with nuclear proteins (Kanedaet al., Science 243 (1989) 375), with lipids (Felgner et al., PNAS 84(1987) 7413), in the form of liposomes (Fraley et al., J. Biol. Chem.255 (1980) 10431), and the like. Preferably, the sequence used withinthe framework of the invention forms part of a vector. The use of such avector indeed makes it possible to improve the administration of thenucleic acid into the cells to be treated, and also to increase itsstability in the said cells, which makes it possible to obtain a lastingtherapeutic effect. Furthermore, it is possible to introduce severalnucleic acid sequences into the same vector, which also increases theefficacy of the treatment.

[0019] The vector used may be of various origin, as long as it iscapable of transforming animal cells, preferably human cancer cells. Ina preferred embodiment of the invention, a viral vector is used whichmay be chosen from adenoviruses, retroviruses, adeno-associated viruses(AAV) or the herpes virus.

[0020] In this regard, the subject of the present invention is anyrecombinant virus comprising, inserted into its genome, a nucleic acidencoding a compound capable of modulating the activity of calpain.Preferably, the viruses used within the framework of the invention aredefective, that is to say that they are incapable of replicatingautonomously in the infected cell. Generally, the genome of thedefective viruses used within the framework of the present inventiontherefore lacks at least the sequences necessary for the replication ofthe said virus in the infected cell. These regions may be either removed(completely or in part), or made nonfunctional, or substituted by othersequences and especially by the sequence encoding the modulator of thecalpains. Preferably, the defective virus retains, nevertheless, thesequences of its genome which are necessary for the encapsidation of theviral particles.

[0021] As regards more particularly adenoviruses, various serotypes,whose structure and properties vary somewhat, have been characterized.Among these serotypes, the use of the type 2 or 5 human adenoviruses (Ad2 or Ad 5) or of the adenoviruses of animal origin (see application FR93 05954) is preferred within the framework of the present invention.Among the adenoviruses of animal origin which can be used within theframework of the present invention, there may be mentioned adenovirusesof canine, bovine, murine (example: MVA1, Beard et al., Virology 75(1990) 81), ovine, porcine, avian or alternatively simian (example: SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, or more preferably a CAV2 adenovirus [Manhattan strain orA26/61 (ATCC VR-800) for example]. Preferably, adenoviruses of human orcanine or mixed origin are used within the framework of the invention.

[0022] Preferably, the defective adenoviruses of the invention comprisethe ITRs, a sequence allowing the encapsidation and the sequenceencoding the modulator of the calpains. Still more preferably, in thegenome of the adenoviruses of the invention, the E1 gene and at leastone of the genes E2, E4, L1-L5 are nonfunctional. The viral geneconsidered can be rendered non-functional by any technique known topersons skilled in the art, and especially by total suppression, bysubstitution or partial deletion, or by addition of one or more bases inthe gene(s) considered. Such modifications can be obtained in vitro (onthe isolated DNA) or in situ, for example by means of geneticengineering techniques, or alternatively by treating with mutagenicagents.

[0023] The defective recombinant adenoviruses according to the inventioncan be prepared by any technique known to persons skilled in the art(Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3(1984) 2917). In particular, they can be prepared by homologousrecombination between an adenovirus and a plasmid carrying, inter alia,the DNA sequence encoding the modulator of the calpains. The homologousrecombination occurs after co-transfection of the said adenoviruses andplasmid into an appropriate cell line. The cell line used shouldpreferably (i) be transformable by the said elements, and (ii) containthe sequences capable of complementing the defective adenovirus genomepart, preferably in integrated form in order to avoid risks ofrecombination. As an example of a cell line, there may be mentioned thehuman embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977)59) which contains especially, integrated in its genome, the left handpart of the genome of an Ad5 adenovirus (12%). Strategies forconstructing vectors derived from adenoviruses have also been describedin Applications Nos. FR 93 05954 and FR 93 08596.

[0024] Next, the adenoviruses which have multiplied are recovered andpurified according to conventional molecular biology techniques asillustrated in the examples.

[0025] As regards the adeno-associated viruses (AAV), they arerelatively small DNA viruses which become integrated into the genome ofthe cells which they infect, in a stable and site-specific manner. Theyare capable of infecting a broad spectrum of cells, without inducing anyeffect on cell growth, morphology or differentiation. Moreover, they donot seem to be involved in pathologies in man. The genome of the AAVshas been cloned, sequenced and characterized. It comprises about 4700bases and contains, at each end, an inverted repeat region (ITR) ofabout 145 bases which serves as replication origin for the virus. Theremainder of the genome is divided into 2 essential regions carrying theencapsidation functions: the left hand part of the genome, whichcontains the rep gene involved in the viral replication and theexpression of the viral genes; the right hand part of the genome, whichcontains the cap gene encoding the virus capsid proteins.

[0026] The use of vectors derived from AAVs for the transfer of genes invitro and in vivo has been described in the literature (see especiallyWO 91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488528). These applications describe various constructs derived from AAVs,from which the rep and/or cap genes are deleted and replaced by a geneof interest, and their use for the transfer in vitro (on cells inculture) or in vivo (directly in an organism) of the said gene ofinterest. The defective recombinant AAVs according to the invention canbe prepared by co-transfection, into a cell line infected by a humanhelper virus (for example an adenovirus), of a plasmid containing thesequence encoding the modulator of the calpains bordered by two AAVinverted repeat regions (ITR), and of a plasmid carrying the AAVencapsidation genes (rep and cap genes). The recombinant AAVs producedare then purified by conventional techniques.

[0027] As regards the herpes viruses and the retroviruses, theconstruction of recombinant vectors has been widely described in theliterature: see especially Breakfield et al., New Biologist 3 (1991)203; EP 453242, EP 178220, Bernstein et al. Genet. Eng. 7 (1985) 235;McCormick, BioTechnology 3 (1985) 689, and the like.

[0028] For carrying out the present invention, it is most particularlyadvantageous to use a defective recombinant retrovirus or adenovirus.These vectors indeed have particularly advantageous properties for thetransfer of genes into tumour cells.

[0029] Advantageously, in the vectors of the invention, the sequenceencoding the modulator of the calpains is placed under the control ofsignals allowing its expression in tumour cells. Preferably, these areheterologous expression signals, that is to say signals different fromthose which are naturally responsible for the expression of themodulator. They may be in particular sequences responsible for theexpression of other proteins, or synthetic sequences. In particular,they may be promoter sequences of eukaryotic or viral genes. Forexample, they may be promoter sequences derived from the genome of thecell which it is desired to infect. Likewise, they may be promotersequences derived from the genome of a virus, including the virus used.In this regard, the E1A, MLP, CMV, RSV-LTR promoters and the like may bementioned for example. In addition, these expression sequences may bemodified by addition of activating or regulatory sequences or ofsequences allowing a tissue-specific expression. It may indeed beparticularly advantageous to use expression signals which are activespecifically or predominantly in tumour cells, so that the DNA sequenceis expressed or produces its effect only when the virus has effectivelyinfected a tumour cell.

[0030] In a specific embodiment, the invention relates to a defectiverecombinant virus comprising a cDNA sequence encoding a modulator of thecalpains under the control of a viral promoter, preferably chosen fromthe RSV-LTR and the CMV promoter.

[0031] Still in a preferred embodiment, the invention relates to adefective recombinant virus comprising a DNA sequence encoding amodulator of the calpains under the control of a promoter allowingpredominant expression in tumour cells.

[0032] The expression is considered to be predominant for the purposesof the invention when, even if a residual expression is observed inother cell types, the expression levels are greater in the tumour cells.

[0033] The present invention also relates to any pharmaceuticalcomposition comprising one or more defective recombinant viruses asdescribed above. These pharmaceutical compositions may be formulated foradministrations via the topical, oral, parenteral, intranasal,intravenous, intramuscular, subcutaneous, intraocular or transdermalroute and the like. Preferably, the pharmaceutical compositions of theinvention contain a vehicle pharmaceutically acceptable for aninjectable formulation, especially for a direct injection into thepatient's tumour. This may be in particular isotonic sterile solutions,or dry, especially freeze-dried, compositions which, upon addition,depending on the case, of sterile water or of physiological saline,allow the preparation of injectable solutions. Direct injection into thepatient's tumour is advantageous because it makes it possible toconcentrate the therapeutic effect at the level of the affected tissues.

[0034] The doses of defective recombinant virus which are used for theinjection may be adapted according to various parameters, and especiallyaccording to the viral vector, the mode of administration used, therelevant pathology or alternatively the desired duration of thetreatment. In general, the recombinant adenoviruses according to theinvention are formulated and administered in the form of doses ofbetween 10⁴ to 10¹⁴ pfu/ml, and preferably 10⁶ to 10¹⁰ pfu/ml. The termpfu (“plaque forming unit”) corresponds to the infectivity of a virussolution, and is determined by infecting an appropriate cell culture andmeasuring, generally after 48 hours, the number of plaques of infectedcells. The techniques for determining the pfu titre of a viral solutionare well documented in the literature. As regards the retroviruses, thecompositions according to the invention may directly comprise theproducing cells, for their implantation.

[0035] The present invention is particularly adapted to the treatment ofcancers in which the mutated forms of p53 are observed. Morespecifically, the present invention is particularly advantageous for thetreatment of cancers in which the wild-type and mutated alleles of p53are present. Such cancers are especially colorectal cancer, breastcancer, lung cancer, gastric cancer, oesophageal cancer, B lymphomas,ovarian cancer, cancer of the bladder and the like.

[0036] The present invention will be more fully described with the aidof the following Examples which should be considered as illustrative andnonlimiting.

LEGEND TO THE FIGURES

[0037]FIG. 1: Study of the regulation of the p53 protein by calpain. Thereaction is carried out in a final volume of 30 μl, of which 1 comesfrom the translation mixture. Line 1: T0; line 2: 30 min in the presenceof 1 mM Calcium+20 μg/ml Calpain; line 4: 30 min in the presence of 1 mMCalcium+20 μg/ml Calpain+0.5 mg/ml calpastatin; line 5: 30 min in thepresence of 1 mM Calcium+20 μg/ml Calpain+10 mM EGTA; line 6: PBS; line7: PBS+calcium; line 8: PBS+calpastatin.

GENERAL MOLECULAR BIOLOGY TECHNIQUES

[0038] The methods conventionally used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA incaesium chloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in saline medium, transformation in Escherichiacoli and the like, are well known to persons skilled in the art and arewidely described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

[0039] The pBR322 and pUC type plasmids and the phages of the M13 seriesare of commercial origin (Bethesda Research Laboratories).

[0040] For the ligations, the DNA fragments can be separated accordingto their size by agarose or acrylamide gel electrophoresis, extractedwith phenol or with a phenol/chloroform mixture, precipitated withethanol and then incubated in the presence of phage T4 DNA ligase(Biolabs) according to the recommendations of the supplier.

[0041] The filling of the protruding 5′ ends can be performed with theKlenow fragment of E. coli DNA polymerase I (Biolabs) according to thespecifications of the supplier. The destruction of the protruding 3′ends is performed in the presence of phage T4 DNA polymerase (Biolabs)used according to the recommendations of the manufacturer. Thedestruction of the protruding 5′ ends is performed by a controlledtreatment with S1 nuclease.

[0042] Site-directed mutagenesis in vitro by syntheticoligodeoxynucleotides can be performed according to the method developedby Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kitdistributed by Amersham.

[0043] The enzymatic amplification of the DNA fragments by the so-calledPCR technique [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al.,Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth.Enzym. 155 (1987) 335-350] can be performed using a DNA-thermal cycler(Perkin Elmer Cetus) according to the specifications of themanufacturer.

[0044] The verification of the nucleotide sequences can be performed bythe method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74(1977) 5463-5467] using the kit distributed by Amersham.

EXAMPLES Example 1

[0045] This example shows that the addition of m-calpain to rabbitreticulocyte lysate induces the degradation of the wild-type p53 proteinas well as that of certain mutated forms. This example also shows thatinhibitors of calpains are capable of inhibiting the degradation of p53and therefore of modulating the activity of this protein.

[0046] 1.1. Demonstration of the degradation: mouse and human wild-typep53 proteins as well as various mutated p53 proteins (human proteinsC273, H273, H175, 1247) were translated in the rabbit reticulocytelysate. The proteins thus obtained are resistant to any degradation,even in the presence of a high concentration of calcium (cofactoressential for the calpains). The addition of bovine m-calpain (Sigma) tothe reticulocyte lysate in the presence of calcium led to the rapiddisappearance of the neosynthesized proteins and the appearance ofproteolytic fragments which are resolvable by electrophoresis. Thedegradation resistance of other proteins such as dihydrofolate reductaseor glyceraldehyde-3-phosphate dehydrogenase under the same experimentalconditions indicates the substrate specificity of the reaction.

[0047] 1.2. Use of inhibitors of calpain for modulating the levels ofp53 proteins: in the above Example 1.1., it was shown that the additionof m-calpain induced degradation of the p53 proteins. In this example,in addition to m-calpain, various compounds were introduced into themedium in order to test their capacity to inhibit the activity ofcalpain. The results obtained show that the addition of a calciumchelator (EGTA) as well as of a peptide which is a specific inhibitor ofthe calpains (derivative of a physiological inhibitor, calpastatin; Makiet al., J. Biol. Chem., 254, 18866-18869, 1989) are capable ofinhibiting the degradation of the p53 proteins which is induced by theexogenous calpain.

Example 2

[0048] In the preceding example, it was shown that the addition ofexogenous calpain to a solution of p53 proteins brought about theirdegradation. This example shows that the degradation of the wild-typep53 protein as well as that of certain mutated forms may be induced bythe endogenous calpains in cytoplasmic extracts. This example also showsthat inhibitors of the calpains are capable, in the presence ofendogenous calpain, of inhibiting the degradation of p53 and thereforeof modulating the activity of this protein.

[0049] 2.1. Degradation by the endogenous calpains: mouse and humanwild-type p53 proteins, as well as certain mutated forms (cf Example 1)were translated in the reticulocyte lysate and were then incubated inthe presence of cytoplasmic extracts of Daudi or Jurkat humanlymphoblastoid cells. The cytoplasmic extracts were prepared in thefollowing manner: the cells (available at the ATCC) were cultured inDMEM medium supplemented with 10% foetal calf serum. The cells were thenharvested, washed in PBS buffer and then incubated for 5 min in adetergent-free hypotonic lysis buffer (HEPES 20 mM, pH 7.5; KOAc 10 mM;MgOAc 1.5 mM; 2 ml per 5×10⁸ cells). The lysis was completed using aDounce homogenizer and then checked under a microscope. The nuclei werethen removed by centrifugation at 2000 g for 5 min, and the supernatantswere centrifuged at 10,000 g for 1 hour (Beckman SW60). The cytoplasmicextracts were then aliquoted in an amount of 5 to 12 mg/ml.

[0050] When the lysate of reticulocutes was incubated in the presence ofcytoplasmic extracts, in the absence of calcium, no degradation wasobserved. On the other hand, in the presence of calcium, a very rapiddegradation of the p53 proteins was observed, with the appearance of acharacteristic proteolytic product profile similar to that obtained inExample 1. This experiment indeed shows that the p53 proteins aredegraded by the endogenous calpains.

[0051] 2.2. Use of calpain inhibitors to modulate the levels of p53proteins: the chelation of calcium by EGTA, as well as the use of awhole range of protease inhibitors (leupeptin, aprotinin, soybeantrypsin inhibitor and PMSF) and especially the peptide calpastatin showthat the degradation of these proteins is dependent on the calpains ofthe cytoplasmic extract, and that various compounds capable ofmodulating the activity of the calpains may be used to regulate the p53protein levels.

Example 3

[0052] This example demonstrates that the mouse and human wild-type p53proteins are direct substrates for the calpains in the cytoplasmicextracts.

[0053] Examples 1 and 2 show that the calpains can induce thedegradation of p53 in complex reaction mixtures. These experiments donot exclude, however, that under the conditions used, the calpainsactivate secondary proteases which are those which actually act on p53.In this example, the following experiment was conducted: (1) the mouseand human wild-type p53 proteins neosynthesized in the rabbitreticulocyte lysate were incubated for 30 minutes in the presence of acytoplasmic extract of Daudi cells as well as in the presence of calciumto activate the calpains as in Example 2, (2) p53 protein was then addedto the reaction mixture and the reaction was continued for 30 minutesunder conditions permissive (same reaction conditions) or otherwise(addition either of EGTA to chelate the calcium, or of calpastatinpeptide) for the calpains. In the presence of calcium, the newly addedp53 protein is completely degraded, indicating that the proteaseactivity is functional throughout the experiment. When the calpains areinhibited by the presence of EGTA or, more significantly, of thecalpastatin peptide, the newly added p53 protein is, on the other hand,no longer degraded. This latter observation therefore excludes thepossibility that in the first part of the experiment, the calpainsinduced a second protease responsible for the degradation of p53 (FIG.1).

Example 4

[0054] This example describes the construction of a recombinantadenovirus comprising a nucleic acid sequence encoding calpastatin. Thisadenovirus is constructed by homologous recombination between thedefective adenovirus Ad-d11324 and a plasmid carrying the sequence SEQID No. 1 under the control of the RSV promoter.

[0055] 4.1. Construction of the Plasmid SEQ ID No. 1

[0056] The plasmid SEQ ID No. 1 comprises the sequence encodingcalpastatin under the control of the RSV-LTR promoter, as well asregions of the adenovirus which allow homologous recombination. It isconstructed by inserting the sequence SEQ ID No. 1 into the plasmidpAd.RSVβgal. The plasmid pAd.RSVβGal contains, in the 5′—>3′orientation,

[0057] the PvuII fragment corresponding to the left hand end of the Ad5adenovirus comprising: the ITR sequence, the replication origin, theencapsidation signals and the enhancer E1A;

[0058] the gene encoding β-galactosidase under the control of the RSVpromoter (Rous sarcoma virus),

[0059] a second fragment of the Ad5 adenovirus genome which allowshomologous recombination between the plasmid pAd.RSVβGal and theadenovirus d1324. The plasmid pAd.RSVβGal has been described byStratford-Perricaudet et al. (J. Clin. Invest. 90 (1992) 626).

[0060] 4.2. Construction of the Recombinant Adenovirus

[0061] The vector described in 4.1. is linearized and cotransfected witha deficient adenoviral vector into the helper cells (line 293) providingin trans the functions encoded by the adenovirus E1 regions (E1A andE1B).

[0062] More specifically, the recombinant adenovirus is obtained byhomologous recombination in vivo between the mutant adenovirus Ad-d11324(Thimmappaya et al., Cell 31 (1982) 543) and the vector described inExample 4.1., according to the following procedure: the plasmid SEQ IDNo. 1 and the adenovirus Ad-d11324, linearized by the enzyme ClaI, arecotransfected into the line 293 in the presence of calcium phosphate, soas to allow the homologous recombination. The recombinant adenovirusesthus generated are then selected by plaque purification. Afterisolation, the recombinant adenovirus DNA is amplified in the cell line293, leading to a culture supernatant containing the unpurifiedrecombinant defective adenovirus having a titre of about 10¹⁰ pfu/ml.

[0063] The viral particles are purified by centrifugation on a caesiumchloride gradient according to known techniques (see especially Grahamet al., Virology 52 (1973) 456). The adenovirus obtained may be storedat −80° C. in 20% glycerol.

1 4 1 2085 DNA Homo sapiens CDS (1)..(2085) 1 atg gaa gga cca cat cttcct aac aag aaa aaa cac aaa aaa cag gct 48 Met Glu Gly Pro His Leu ProAsn Lys Lys Lys His Lys Lys Gln Ala 1 5 10 15 gta aaa aca gaa cct gagaag aag tca cag tca acc aag ctg tct gtg 96 Val Lys Thr Glu Pro Glu LysLys Ser Gln Ser Thr Lys Leu Ser Val 20 25 30 gtt cat gag aaa aaa tcc caagaa gga aag cca aaa gaa cac aca gag 144 Val His Glu Lys Lys Ser Gln GluGly Lys Pro Lys Glu His Thr Glu 35 40 45 cca aaa agc cta ccc aag cag gcatca gat aca gga agt aac gat gct 192 Pro Lys Ser Leu Pro Lys Gln Ala SerAsp Thr Gly Ser Asn Asp Ala 50 55 60 cac aat aaa aaa gca gtt tcc aga tcagct gaa cag cag cca tca gag 240 His Asn Lys Lys Ala Val Ser Arg Ser AlaGlu Gln Gln Pro Ser Glu 65 70 75 80 aaa tca aca gaa cca aag act aaa ccacaa gac atg att tct gct ggt 288 Lys Ser Thr Glu Pro Lys Thr Lys Pro GlnAsp Met Ile Ser Ala Gly 85 90 95 gga gag agt gtt gct ggt atc act gca atatct ggc aag ccg ggt gac 336 Gly Glu Ser Val Ala Gly Ile Thr Ala Ile SerGly Lys Pro Gly Asp 100 105 110 aag aaa aaa gaa aag aaa tca tta acc ccagct gtg cca gtt gaa tct 384 Lys Lys Lys Glu Lys Lys Ser Leu Thr Pro AlaVal Pro Val Glu Ser 115 120 125 aaa ccg gat aaa cca tcg gga aag tca ggcatg gat gct gct ttg gat 432 Lys Pro Asp Lys Pro Ser Gly Lys Ser Gly MetAsp Ala Ala Leu Asp 130 135 140 gac tta ata gat act tta gga gga cct gaagaa act gaa gaa gaa aat 480 Asp Leu Ile Asp Thr Leu Gly Gly Pro Glu GluThr Glu Glu Glu Asn 145 150 155 160 aca acg tat act gga cca gaa gtt tcagat cca atg agt tcc acc tac 528 Thr Thr Tyr Thr Gly Pro Glu Val Ser AspPro Met Ser Ser Thr Tyr 165 170 175 ata gag gaa ttg ggt aaa aga gaa gtcaca att cct cca aaa tat agg 576 Ile Glu Glu Leu Gly Lys Arg Glu Val ThrIle Pro Pro Lys Tyr Arg 180 185 190 gaa cta ttg gct aaa aag gaa ggg atcaca ggg cct cct gca gac tct 624 Glu Leu Leu Ala Lys Lys Glu Gly Ile ThrGly Pro Pro Ala Asp Ser 195 200 205 tca aaa ccc ata ggg cca gat gat gctata gac gcc ttg tca tct gac 672 Ser Lys Pro Ile Gly Pro Asp Asp Ala IleAsp Ala Leu Ser Ser Asp 210 215 220 ttc acc tgt ggg tcg cct aca gct gctgga aag aaa act gaa aaa gag 720 Phe Thr Cys Gly Ser Pro Thr Ala Ala GlyLys Lys Thr Glu Lys Glu 225 230 235 240 gaa tct aca gaa gtt tta aaa gctcag tca gca ggg aca gtc aga agt 768 Glu Ser Thr Glu Val Leu Lys Ala GlnSer Ala Gly Thr Val Arg Ser 245 250 255 gct gct cca ccc caa gag aag aaaaga aag gtg gag aag gat aca atg 816 Ala Ala Pro Pro Gln Glu Lys Lys ArgLys Val Glu Lys Asp Thr Met 260 265 270 agt gat caa gca ctc gag gct ctgtcg gct tca ctg ggc acc cgg caa 864 Ser Asp Gln Ala Leu Glu Ala Leu SerAla Ser Leu Gly Thr Arg Gln 275 280 285 gca gaa cct gag ctc gac ctc cgctca att aag gaa gtc gat gag gca 912 Ala Glu Pro Glu Leu Asp Leu Arg SerIle Lys Glu Val Asp Glu Ala 290 295 300 aaa gct aaa gaa gaa aaa cta gagaag tgt ggt gag gat gat gaa aca 960 Lys Ala Lys Glu Glu Lys Leu Glu LysCys Gly Glu Asp Asp Glu Thr 305 310 315 320 atc cca tct gag tac aga ttaaaa cca gcc acg gat aaa gat gga aaa 1008 Ile Pro Ser Glu Tyr Arg Leu LysPro Ala Thr Asp Lys Asp Gly Lys 325 330 335 cca cta ttg cca gag cct gaagaa aaa ccc aag cct cgg agt gaa tca 1056 Pro Leu Leu Pro Glu Pro Glu GluLys Pro Lys Pro Arg Ser Glu Ser 340 345 350 gaa ctc att gat gaa ctt tcagaa gat ttt gac cgg tct gaa tgt aaa 1104 Glu Leu Ile Asp Glu Leu Ser GluAsp Phe Asp Arg Ser Glu Cys Lys 355 360 365 gag aaa cca tct aag cca actgaa aag aca gaa gaa tct aag gcc gct 1152 Glu Lys Pro Ser Lys Pro Thr GluLys Thr Glu Glu Ser Lys Ala Ala 370 375 380 gct cca gct cct gtg tcg gaggct gtg tct cgg acc tcc atg tgt agt 1200 Ala Pro Ala Pro Val Ser Glu AlaVal Ser Arg Thr Ser Met Cys Ser 385 390 395 400 ata cag tca gca ccc cctgag ccg gct acc ttg aag ggc aca gtg cca 1248 Ile Gln Ser Ala Pro Pro GluPro Ala Thr Leu Lys Gly Thr Val Pro 405 410 415 gat gat gct gta gaa gccttg gct gat agc ctg ggg aaa aag gaa gca 1296 Asp Asp Ala Val Glu Ala LeuAla Asp Ser Leu Gly Lys Lys Glu Ala 420 425 430 gat cca gaa gat gga aaacct gtg atg gat aaa gtc aag gag aag gcc 1344 Asp Pro Glu Asp Gly Lys ProVal Met Asp Lys Val Lys Glu Lys Ala 435 440 445 aaa gaa gaa gac cgt gaaaag ctt ggt gaa aaa gaa gaa aca att cct 1392 Lys Glu Glu Asp Arg Glu LysLeu Gly Glu Lys Glu Glu Thr Ile Pro 450 455 460 cct gat tat aga tta gaagag gtc aag gat aaa gat gga aag cca ctc 1440 Pro Asp Tyr Arg Leu Glu GluVal Lys Asp Lys Asp Gly Lys Pro Leu 465 470 475 480 ctg cca aaa gag tctaag gaa cag ctt cca ccc atg agt gaa gac ttc 1488 Leu Pro Lys Glu Ser LysGlu Gln Leu Pro Pro Met Ser Glu Asp Phe 485 490 495 ctt ctg gat gct ttgtct gag gac ttc tct ggt cca caa aat gct tca 1536 Leu Leu Asp Ala Leu SerGlu Asp Phe Ser Gly Pro Gln Asn Ala Ser 500 505 510 tct ctt aaa ttt gaagat gct aaa ctt gct gct gcc atc tct gaa gtg 1584 Ser Leu Lys Phe Glu AspAla Lys Leu Ala Ala Ala Ile Ser Glu Val 515 520 525 gtt tcc caa acc ccagct tca acg acc caa gct gga gcc cca ccc cgt 1632 Val Ser Gln Thr Pro AlaSer Thr Thr Gln Ala Gly Ala Pro Pro Arg 530 535 540 gat acc tcg cag agtgac aaa gac ctc gat gat gcc ttg gat aaa ctc 1680 Asp Thr Ser Gln Ser AspLys Asp Leu Asp Asp Ala Leu Asp Lys Leu 545 550 555 560 tct gac agt ctagga caa agg cag cct gac cca gat gag aac aaa cca 1728 Ser Asp Ser Leu GlyGln Arg Gln Pro Asp Pro Asp Glu Asn Lys Pro 565 570 575 atg gga gat aaagta aag gaa aaa gct aaa gct gaa cat aga gac aag 1776 Met Gly Asp Lys ValLys Glu Lys Ala Lys Ala Glu His Arg Asp Lys 580 585 590 ctt gga gaa agagat gac act atc cca cct gaa tac aga cat ctc ctg 1824 Leu Gly Glu Arg AspAsp Thr Ile Pro Pro Glu Tyr Arg His Leu Leu 595 600 605 gat gat aat ggacag gac aaa cca gtg aag cca cct aca aag aaa tca 1872 Asp Asp Asn Gly GlnAsp Lys Pro Val Lys Pro Pro Thr Lys Lys Ser 610 615 620 gag gat tca aagaaa cct gca gat gac caa gac ccc att gat gct ctc 1920 Glu Asp Ser Lys LysPro Ala Asp Asp Gln Asp Pro Ile Asp Ala Leu 625 630 635 640 tca gga gatctg gac agc tgt ccc tcc act aca gaa acc tca cag aac 1968 Ser Gly Asp LeuAsp Ser Cys Pro Ser Thr Thr Glu Thr Ser Gln Asn 645 650 655 aca gca aaggat aag tgc aag aag gct gct tcc agc tcc aaa gca cct 2016 Thr Ala Lys AspLys Cys Lys Lys Ala Ala Ser Ser Ser Lys Ala Pro 660 665 670 aag aat ggaggt aaa gcg aag gat tca gca aag aca aca gag gaa act 2064 Lys Asn Gly GlyLys Ala Lys Asp Ser Ala Lys Thr Thr Glu Glu Thr 675 680 685 tcc aag ccaaaa gat gac taa 2085 Ser Lys Pro Lys Asp Asp 690 695 2 694 PRT Homosapiens 2 Met Glu Gly Pro His Leu Pro Asn Lys Lys Lys His Lys Lys GlnAla 1 5 10 15 Val Lys Thr Glu Pro Glu Lys Lys Ser Gln Ser Thr Lys LeuSer Val 20 25 30 Val His Glu Lys Lys Ser Gln Glu Gly Lys Pro Lys Glu HisThr Glu 35 40 45 Pro Lys Ser Leu Pro Lys Gln Ala Ser Asp Thr Gly Ser AsnAsp Ala 50 55 60 His Asn Lys Lys Ala Val Ser Arg Ser Ala Glu Gln Gln ProSer Glu 65 70 75 80 Lys Ser Thr Glu Pro Lys Thr Lys Pro Gln Asp Met IleSer Ala Gly 85 90 95 Gly Glu Ser Val Ala Gly Ile Thr Ala Ile Ser Gly LysPro Gly Asp 100 105 110 Lys Lys Lys Glu Lys Lys Ser Leu Thr Pro Ala ValPro Val Glu Ser 115 120 125 Lys Pro Asp Lys Pro Ser Gly Lys Ser Gly MetAsp Ala Ala Leu Asp 130 135 140 Asp Leu Ile Asp Thr Leu Gly Gly Pro GluGlu Thr Glu Glu Glu Asn 145 150 155 160 Thr Thr Tyr Thr Gly Pro Glu ValSer Asp Pro Met Ser Ser Thr Tyr 165 170 175 Ile Glu Glu Leu Gly Lys ArgGlu Val Thr Ile Pro Pro Lys Tyr Arg 180 185 190 Glu Leu Leu Ala Lys LysGlu Gly Ile Thr Gly Pro Pro Ala Asp Ser 195 200 205 Ser Lys Pro Ile GlyPro Asp Asp Ala Ile Asp Ala Leu Ser Ser Asp 210 215 220 Phe Thr Cys GlySer Pro Thr Ala Ala Gly Lys Lys Thr Glu Lys Glu 225 230 235 240 Glu SerThr Glu Val Leu Lys Ala Gln Ser Ala Gly Thr Val Arg Ser 245 250 255 AlaAla Pro Pro Gln Glu Lys Lys Arg Lys Val Glu Lys Asp Thr Met 260 265 270Ser Asp Gln Ala Leu Glu Ala Leu Ser Ala Ser Leu Gly Thr Arg Gln 275 280285 Ala Glu Pro Glu Leu Asp Leu Arg Ser Ile Lys Glu Val Asp Glu Ala 290295 300 Lys Ala Lys Glu Glu Lys Leu Glu Lys Cys Gly Glu Asp Asp Glu Thr305 310 315 320 Ile Pro Ser Glu Tyr Arg Leu Lys Pro Ala Thr Asp Lys AspGly Lys 325 330 335 Pro Leu Leu Pro Glu Pro Glu Glu Lys Pro Lys Pro ArgSer Glu Ser 340 345 350 Glu Leu Ile Asp Glu Leu Ser Glu Asp Phe Asp ArgSer Glu Cys Lys 355 360 365 Glu Lys Pro Ser Lys Pro Thr Glu Lys Thr GluGlu Ser Lys Ala Ala 370 375 380 Ala Pro Ala Pro Val Ser Glu Ala Val SerArg Thr Ser Met Cys Ser 385 390 395 400 Ile Gln Ser Ala Pro Pro Glu ProAla Thr Leu Lys Gly Thr Val Pro 405 410 415 Asp Asp Ala Val Glu Ala LeuAla Asp Ser Leu Gly Lys Lys Glu Ala 420 425 430 Asp Pro Glu Asp Gly LysPro Val Met Asp Lys Val Lys Glu Lys Ala 435 440 445 Lys Glu Glu Asp ArgGlu Lys Leu Gly Glu Lys Glu Glu Thr Ile Pro 450 455 460 Pro Asp Tyr ArgLeu Glu Glu Val Lys Asp Lys Asp Gly Lys Pro Leu 465 470 475 480 Leu ProLys Glu Ser Lys Glu Gln Leu Pro Pro Met Ser Glu Asp Phe 485 490 495 LeuLeu Asp Ala Leu Ser Glu Asp Phe Ser Gly Pro Gln Asn Ala Ser 500 505 510Ser Leu Lys Phe Glu Asp Ala Lys Leu Ala Ala Ala Ile Ser Glu Val 515 520525 Val Ser Gln Thr Pro Ala Ser Thr Thr Gln Ala Gly Ala Pro Pro Arg 530535 540 Asp Thr Ser Gln Ser Asp Lys Asp Leu Asp Asp Ala Leu Asp Lys Leu545 550 555 560 Ser Asp Ser Leu Gly Gln Arg Gln Pro Asp Pro Asp Glu AsnLys Pro 565 570 575 Met Gly Asp Lys Val Lys Glu Lys Ala Lys Ala Glu HisArg Asp Lys 580 585 590 Leu Gly Glu Arg Asp Asp Thr Ile Pro Pro Glu TyrArg His Leu Leu 595 600 605 Asp Asp Asn Gly Gln Asp Lys Pro Val Lys ProPro Thr Lys Lys Ser 610 615 620 Glu Asp Ser Lys Lys Pro Ala Asp Asp GlnAsp Pro Ile Asp Ala Leu 625 630 635 640 Ser Gly Asp Leu Asp Ser Cys ProSer Thr Thr Glu Thr Ser Gln Asn 645 650 655 Thr Ala Lys Asp Lys Cys LysLys Ala Ala Ser Ser Ser Lys Ala Pro 660 665 670 Lys Asn Gly Gly Lys AlaLys Asp Ser Ala Lys Thr Thr Glu Glu Thr 675 680 685 Ser Lys Pro Lys AspAsp 690 3 399 DNA Homo sapiens CDS (1)..(399) 3 tca ggc atg gat gct gctttg gat gac tta ata gat act tta gga gga 48 Ser Gly Met Asp Ala Ala LeuAsp Asp Leu Ile Asp Thr Leu Gly Gly 1 5 10 15 cct gaa gaa act gaa gaagaa aat aca acg tat act gga cca gaa gtt 96 Pro Glu Glu Thr Glu Glu GluAsn Thr Thr Tyr Thr Gly Pro Glu Val 20 25 30 tca gat cca atg agt tcc acctac ata gag gaa ttg ggt aaa aga gaa 144 Ser Asp Pro Met Ser Ser Thr TyrIle Glu Glu Leu Gly Lys Arg Glu 35 40 45 gtc aca att cct cca aaa tat agggaa cta ttg gct aaa aag gaa ggg 192 Val Thr Ile Pro Pro Lys Tyr Arg GluLeu Leu Ala Lys Lys Glu Gly 50 55 60 atc aca ggg cct cct gca gac tct tcaaaa ccc ata ggg cca gat gat 240 Ile Thr Gly Pro Pro Ala Asp Ser Ser LysPro Ile Gly Pro Asp Asp 65 70 75 80 gct ata gac gcc ttg tca tct gac ttcacc tgt ggg tcg cct aca gct 288 Ala Ile Asp Ala Leu Ser Ser Asp Phe ThrCys Gly Ser Pro Thr Ala 85 90 95 gct gga aag aaa act gaa aaa gag gaa tctaca gaa gtt tta aaa gct 336 Ala Gly Lys Lys Thr Glu Lys Glu Glu Ser ThrGlu Val Leu Lys Ala 100 105 110 cag tca gca ggg aca gtc aga agt gct gctcca ccc caa gag aag aaa 384 Gln Ser Ala Gly Thr Val Arg Ser Ala Ala ProPro Gln Glu Lys Lys 115 120 125 aga aag gtg gag aag 399 Arg Lys Val GluLys 130 4 133 PRT Homo sapiens 4 Ser Gly Met Asp Ala Ala Leu Asp Asp LeuIle Asp Thr Leu Gly Gly 1 5 10 15 Pro Glu Glu Thr Glu Glu Glu Asn ThrThr Tyr Thr Gly Pro Glu Val 20 25 30 Ser Asp Pro Met Ser Ser Thr Tyr IleGlu Glu Leu Gly Lys Arg Glu 35 40 45 Val Thr Ile Pro Pro Lys Tyr Arg GluLeu Leu Ala Lys Lys Glu Gly 50 55 60 Ile Thr Gly Pro Pro Ala Asp Ser SerLys Pro Ile Gly Pro Asp Asp 65 70 75 80 Ala Ile Asp Ala Leu Ser Ser AspPhe Thr Cys Gly Ser Pro Thr Ala 85 90 95 Ala Gly Lys Lys Thr Glu Lys GluGlu Ser Thr Glu Val Leu Lys Ala 100 105 110 Gln Ser Ala Gly Thr Val ArgSer Ala Ala Pro Pro Gln Glu Lys Lys 115 120 125 Arg Lys Val Glu Lys 130

1. Use of a compound capable of modulating the activity of calpain forthe preparation of a pharmaceutical composition for the treatment ofcancer.
 2. Use according to claim 1, characterized in that the compoundis a protein or a polypeptide which is an inhibitor of the activity ofcalpain, or a nucleic acid sequence encoding such a polypeptide orprotein.
 3. Use according to claim 2, characterized in that the compoundis a protein or a polypeptide which is a specific inhibitor of theactivity of calpain on the wild-type p53 protein, or a nucleic acidsequence encoding such a polypeptide or protein.
 4. Use according toclaim 2 or 3, characterized in that the nucleic acid is part of avector.
 5. Use according to claim 4, characterized in that the nucleicacid is part of a viral vector, chosen from adenoviruses, retrovirusesand adeno-associated viruses.
 6. Use according to claim 4, characterizedin that the nucleic acid is part of a lipid liposomal vector.
 7. Useaccording to one of the preceding claims, characterized in that thecompound is a nucleic acid encoding all or part of calpastatin.
 8. Useaccording to claim 7, characterized in that the nucleic acid comprisesall or part of the sequence SEQ ID No. 1 or a derivative thereof.
 9. Useaccording to claim 8, characterized in that the nucleic acid is chosenfrom the sequences SEQ ID No. 1 and
 2. 10. Use according to claim 8,characterized in that the nucleic acid is chosen from the derivatives ofthe sequences SEQ ID No. 1 or 2 encoding specific inhibitors of thedegradation of the wild-type p53 protein.
 11. Use according to one ofclaims 1 to 6, characterized in that the compound is a derivative ofcalpain capable of specifically degrading the mutated p53 proteins. 12.Viral vector comprising a nucleic acid sequence encoding a protein or apolypeptide which is an inhibitor of the activity of calpain.
 13. Vectoraccording to claim 12, characterized in that it is chosen from theadenoviruses, retroviruses and adeno-associated viruses.
 14. Vectoraccording to either of claims 12 or 13, characterized in that itcomprises a sequence encoding all or part of calpastatin.
 15. Vectoraccording to claim 12, characterized in that it comprises a sequenceencoding a derivative of calpain capable of specifically degrading themutated p53 proteins.
 16. Pharmaceutical composition comprising anucleic acid sequence encoding all or part of calpastatin or aderivative of calpain capable of specifically degrading the mutated p53proteins.
 17. Composition according to claim 16, formulated forinttra-tumour administration.